AIM:
To evaluate measurements of intragastric pH with the Bravo capsule
system over a prolonged time.

METHODS:
A Bravo capsule was placed inside the rat gastric body and pH was
studied for periods up to five consecutive days. For comparison, a
gastric fistula model was used. Effects of ghrelin and esomeprazole,
with or without pentagastrin, on gastric pH were studied. In addition,
effects of esomeprazole on plasma ghrelin, gastrin and somatostatin were
analyzed.

CONCLUSION: pH measurements with the Bravo capsule are reliable, and
comparable to those of the gastric fistula model. The Bravo system
optimizes accurate intragastric pH monitoring over prolonged periods and
allows both short- and long-term evaluation of effects of drugs and
hormones.

In the past, different techniques have been employed to
study gastric acid secretion in rodents. The main principle for these
methods has been collection of gastric juice, and in order to measure
acid secretion, pH titration has been carried out. One of the earliest
methods was the pylorus ligation technique[1]. The principle
of this method is distension of the stomach as a potent stimulus of acid
secretion. Later, this method was altered with an esophageal ligature[2,3],
after which the stomach of the rat was removed and secretions analyzed.
Esophageal ligation in the pylorus-ligated rat has been shown to
significantly inhibit acid secretion by inhibition of central vagus
function[2]. Since then, the most reliable method has been
the chronic fistula method[4-6] where a gastric fistula is
implanted at the greater curvature of the stomach. This technique
requires movement restriction of the animal which is in a conscious
state during the study. The gastric contents are collected and acid
output measured. This technique allows re-use of animals following a
recovery period from the experimental procedure. Other methods used
today are perfusion of the gastric lumen[7] and isolated
perfused, as well as vascularly perfused rat stomach[8-10].

Most of the above studies have the drawback that they do
not measure intragastric pH directly and are not very physiological, as
the animal is either restrained or anesthetized. The main goal of this
study was to test the feasibility of a capsule normally used in the
clinical setting in humans to measure gastroesophageal reflux disease
(Bravo system) for monitoring intragastric pH in the rat. The Bravo
capsule system has primarily been used in humans[11-14], but
also in animals[15] for diagnosis of gastroesophageal reflux
disease.

The aim of the study was to evaluate the Bravo capsule
for pH monitoring in the rat. To validate the method, we compared the
data to those of the standard gastric fistula model.

MATERIALS AND METHODS

Animals

Sprague-Dawley male rats (300-350 g) were purchased from
Scanbur B&K AB (Sollentuna, Sweden). The rats were housed in wire-meshed
cages at 24℃
with constant humidity and 12:12 h light-dark cycle. The animals were
fed ad libitum with a commercial rat diet consisting of pellet (LABFOR,
Lactamin R36, Kimstad, Sweden) and tap water prior to the studies. The
experiments were approved by the Animal Ethics Committee in northern
Stockholm.

For the Bravo system studies, a midline incision was
performed, and a small opening created in the proximal greater
curvature, and gastric contents were evacuated. An externally
pre-calibrated (buffers pH 1.07 and 7.1) Bravo capsule (an electronic
sensor encapsulated in PVC-plastic, 25 mm × 5 mm × 5 mm; Synmed
Medicinteknik AB, Spånga, Sweden) was placed inside the stomach with the
pH sensor pointing distally and anchored with a suture. An indwelling
silastic catheter (Dow Corning Co., Midland, MI, USA) was inserted into
the external jugular vein.

For the gastric acid fistula studies, rats were provided
with a plastic gastric fistula placed immediately proximal to the
oxyntic gland area near the greater curvature. The fistula was closed
between experimental periods. A silastic catheter was implanted into the
external jugular vein for drug administration.

Studies of intragastric pH (Bravo system)

Studies of intragastric pH began in the morning 2 d after
surgery. The studies were carried out in conscious rats, one experiment
for each rat, under normal conditions, or after a 16-h fasting period in
wire-bottom cages with free access to water. The animals gained weight
(10 ± 3.4 g during 1 wk) and behaved in a normal fashion, with a normal
feeding pattern throughout the experiments. At post-mortem examination,
no mucosal lesions, obstruction of the pylorus or gastric distension
were seen. Drugs were administered through the external jugular vein in
all experiments.

The pH recorded by the Bravo capsule was transmitted to
the Bravo receiver placed directly outside the cage. The sampling
frequency was 6 Hz. The Bravo system was set for a 48-h registration
period, after which the data were downloaded, batteries replaced and
recording continued. This procedure was then repeated in two more 48-h
periods.

The effect of pentagastrin and esomeprazole on pH: The effect
of esomeprazole (3 mg/kg iv, n = 10) or saline (iv, n = 8)
was studied under pentagastrin (NeoMPS, Strasbourg, France) infusion (90
pmol/kg per min, iv) over 6 h in both fed and fasting rats. In
these experiments, the rats were restrained in Bollman cages to mimic
the gastric fistula studies and for infusion of pentagastrin.

Plasma
levels of gut hormones:
The effect of esomeprazole (3 mg/kg iv) on plasma levels of ghrelin,
gastrin and somatostatin was studied. A group of animals (n = 10)
was divided into two treatment groups (each n = 5). All animals
were treated with esomeprazole daily during 1 wk. The first group of
animals was then euthanized, while the other group was followed for
another week without esomeprazole and then euthanaized. Blood was drawn
and centrifuged, and plasma assayed for concentrations of ghrelin,
gastrin and somatostatin.

For
ghrelin measurements, the ghrelin (active) radioimmunoassay kit (Linco
Research, St. Charles, MI, USA) was used, which utilizes 125I-labeled
ghrelin and ghrelin antiserum to determine the level of active ghrelin
in plasma. For the analysis, a Gamma Master 1277 (LKB-Wallac, Perkin-Elmer
Inc, Massachusetts, NH, USA) was used. The intra- and interassay
coefficients of variation were 7% and 14%, respectively.

Somatostatin was analyzed using an EIA kit (EK-060-03) from Phoenix
Pharmaceuticals, Burlingame, CA, USA), which reacts 100% to
somatostatin-14 and somatostatin-28. The intra- and interassay
coefficients of variation were 5% and 14%, respectively.

Gastrin
was analyzed using C-terminal-directed CCK/gastrin antiserum 2609/10 (Rehfeld,
1978). Chloramine-T-labeled and HPLC-purified gastrin-17 (NeoMPS) was
used as radioligand and gastrin-17 as calibrator/standard. The intra-
and interassay coefficients of variation were 6% and 8%, respectively.

Studies of gastric acid secretion (fistula)

Studies
of gastric acid secretion began 7 d after surgery. The animals gained
weight (8 ± 2.6 g during 1 wk) and had normal behavior during the
experimentation periods. Prior to each experiment, food was withheld for
18 h, but with free access to water. At the start of the experiments,
the stomach was rinsed with 10-15 mL luke-warm tap water to evacuate
remaining food, followed by a 30-min period before the experiments were
started. During the experiments, the conscious rats were placed in
Bollman cages. Gastric juice was collected at 30-min intervals, and
volumes measured to the nearest 0.1 mL. pH was calculated by
back-titration using 0.1 mmol/L sodium hydroxide. Acid output was
calculated by multiplying the secretion volumes with hydrogen ion
concentrations and expressed as mmol per 30-min period.

Baseline
acid secretion was studied for 60 min followed by esomeprazole (3 mg/kg
iv), after which acid secretion was studied for another 2 h. During the
experiment, saline was administered in the same amount as collected from
the gastric fistula to compensate for the volume loss during the
experiment. Furthermore, baseline acid secretion was studied for 60 min,
followed by an infusion of pentagastrin (90 pmol/kg per min) for 4 h.
After 1 h of pentagastrin infusion, a bolus of esomeprazole (3 mg/kg iv)
was administered and acid secretion studied for another 3 h.

Data and statistical analysis

The data obtained with the Bravo capsule analyzed using
(POLYGRAM NET™ pH Testing Application software, Synmed Medicinteknik) in
48-h periods. Results of studies with esomeprazole were analyzed by
calculating changes in pH at various timepoints from baseline (defined
as 0.5 h prior to onset of studies). For analysis of the fistula
studies, the first 30-min collection was discarded and the second
collection used as baseline for comparison with esomeprazole and
pentagastrin.

All data
are mean ± SE. A Kruskal-Wallis test followed by Mann-Whitney U
test was used for statistical comparisons using specific time points for
pH. P < 0.05 was considered statistically significant. For
comparison of the variability between the fistula and the Bravo system
the Bland-Altman analysis was used[16,17]. The Prism software
package 4.0 (GraphPad Software Inc., San Diego, CA, USA) was used for
the statistical comparisons.

RESULTS

Comparison between the Bravo system and the fistula model

Pentagastrin resulted in a marked increase, 83 ± 9 mmol/L to 132 ± 8
mmol/L (P < 0.05) of acid output in the fistula model, which was
not evident as a corresponding decrease in pH with the Bravo system.
During esomeprazole treatment, there was a marked increase in pH from
2.0 ± 0.2 to 3.7 ± 0.5, as recorded with the Bravo system and
correspondingly, a marked decrease in acid secretion from 105 ± 21 mmol/L
to 31 ± 7 mmol/L in the fistula model (P < 0.05; Figure 1).
Bland-Altman analysis of these conditions showed a high degree of
agreement between the Bravo system and the fistula method as shown in
Figure 2.

Evaluation of basal pH

A typical
120-h baseline registration including dose of esomeprazole (day 1, 3 and
5) with the Bravo system is shown in Figure 3. The feeding status did
not alter the mean pH over 24 h, but increases in pH were observed
during afternoon and night-time when animals were fed. The mean 24-h pH
was 2.3 ± 0.1 during fed conditions and 2.5 ± 0.3 during fasted
conditions, with 18% ± 6% variation during the next four 24-h periods.
There was no difference in pH between daytime and night-time (1.4 ± 0.1
and 1.7 ± 0.2, respectively).

After
esomeprazole (9 pmol/kg per min) the average 24-h pH was substantially
higher than in the controls, 5.7 ± 0.3 and 2.1 ± 0.2, respectively (P
< 0.01). Pentagastrin alone did not change pH over the 24-h infusion
period as compared to saline (Figure 6).

The
effect of ghrelin on pH

Administration of ghrelin, t.i.d markedly increased gastric 24-h pH from
day 1 (2.5 ± 0.6) to day 5 (2.8 ± 0.5) compared to control day 1 (1.4 ±
0.1) and day 5 (1.5 ± 0.2) (P < 0.01; n = 7). There was no
significant day-to-day variation of the ghrelin effect during the five
days (Figure 7).

Plasma levels of gut hormones

Esomeprazole (3 mg/kg) t.i.d resulted in a marked
increase in plasma ghrelin and somatostatin concentrations as shown in
Figure 8 (P < 0.001). Plasma gastrin, however, remained stable
over the same time period (Figure 8).

DISCUSSION

This
study demonstrates that the Bravo system can be used for studies of
intragastric pH in rats and that the results are comparable to those of
a standard fistula model. The system allows for long-term studies during
unrestrained living conditions. There are several advantages with the
use of the Bravo system. Previous models for studies of gastric acid
secretion do not allow measurements of pH over a long time. Furthermore,
during these studies, the animals are kept under stressful conditions,
which to a certain degree, may influence the responsiveness of the
animals to different stimuli. The Bravo system uses a telemetric system
that records gastric pH during 24 h for up to five consecutive days. The
day-to-day variation was within acceptable limits. The system allows for
real-time recordings of intragastric pH with the ability to record from
the start of a treatment until a detectable effect is seen. The system
is suitable for long-term studies with continuous infusions that are
difficult to perform using the fistula model, as the animals do not
tolerate being restrained in cages during prolonged studies. The Bravo
recording system is also a digital recording system, which means that
primary data are logged, and permits detailed measurements as determined
by the set sampling frequency.

The data
are, however, limited to pH-values as no secretion volumes are obtained.
With the gastric fistula model, recordings are made over no less than
15-min periods, which can be a limiting factor as regards rapid changes
in pH, i.e. drug effects. However, in the fistula model, secretion
volumes are recorded, which permit calculation of a true acid output.
The Bravo system has a few drawbacks. It is expensive, the battery life
of the capsule is short (5 d) and therefore, the animals can only be
used in studies for about a week. This means that experiments must start
immediately after the operation (in this case 2 d after the surgical
procedure), and the recovery from surgery may influence the results and
the comparison with the fistula model. Despite this, the Bravo system
seems to be well tolerated, as the stomach of the rats did not show any
abnormalities or mucosal lesions upon autopsy. The animals also gain
weight and behaved in a normal fashion during the experiments.

From a
physiological viewpoint, our results demonstrated expected results;
intragastric pH in rodents was stable over time, with a slight increase
during the night during fed conditions.

In
addition, treatment with esomeprazole and pentagastrin gave expected
results. The agreement between the Bravo system and the established
fistula method was evaluated employing a Bland-Altman analysis. When the
two methods were compared, the pH results obtained with the Bravo system
were comparable to those obtained using the fistula model. The
differences lie within acceptable limits of agreement approximately 95%
of the time, and the variability was consistent across the graph; the
scatter around the baseline (mean) did not increase with increasing
means.

During
comparative studies, the animals were restrained in Bollman cages for
infusion of pentagastrin, so the experimental conditions were the same.
During esomeprazole treatment, pH rose and gastric acid output decreased
accordingly. There seemed to be a slight delay in response to
esomeprazole when studied by the fistula method as compared to the Bravo
system. The reason for this is probably related to the fact that the
secretory response depends on the physical emptying of gastric contents
from the fistula until measurements can be done. As judged from our
experiments, this causes a delay of the recorded response of about 30
min. Pentagastrin increased acid output, but no change was seen in
intragastric pH with the Bravo system. This is explained by the fact
that a change in secretion volume does not affect the pH recorded, even
though acid output is changed. The fact that pH does not change when
introducing pentagastrin may be due to the constantly low basal pH level
in the rat stomach.

The gut
hormones assayed in this study, ghrelin, gastrin, and somatostatin, are
all found in the mucosa of the stomach[18]. They operate in a
coherent inhibitory/stimulatory fashion against one another, i.e.
increasing levels of somatostatin stimulates ghrelin, while gastrin is
inhibited[19,20]. Pentagastrin acts as an agonist on acid
secretion and has a stimulatory effect on somatostatin, which in turn
down-regulates the release of gastrin so that excessive amounts of acid
are not produced[20]. The fact that basal plasma gastrin
levels remained stable with the Bravo system indicates that the Bravo
capsule by itself does not distend the stomach to such a degree that
gastrin levels are affected[21].

Our
results using the Bravo system, with an increase of intragastric pH
during 1 wk after three times daily, administration of ghrelin, are in
accordance with earlier studies[19,22], but at variance with
another[23]. This may be explained by the fact that different
methods for studying gastric acid secretion have been employed, some of
which are dependent on gastric motility for the emptying of gastric
secretions through the fistula. By using the Bravo system, we found no
desensitization of the pH response to ghrelin. This is at variance with
our previous studies on intestinal motility, in which a loss of the
ghrelin response was shown[19,24]. This might be due to the
fact that motility was stimulated by a continuous infusion of the
hormone, whereas the pH effect was brought about by repeated injections
of ghrelin, a form of administration that is considered less liable to
desensitization effects. As ghrelin not only increases intragastric pH,
but also stimulates gastric emptying in rodents[22,23,25,26].
This may be an erroneous factor in determining acid secretion using the
fistula method.

With
esomeprazole treatment, plasma concentrations of ghrelin and
somatostatin were increased. This effect was maintained for 1 wk after
esomeprazole treatment. The underlying mechanism for this increase in
plasma ghrelin and somatostatin is not yet fully understood, but may be
due to a direct effect of esomeprazole on ghrelin and somatostatin, but
also by an indirect effect through changes in gastric pH. The
counter-balancing effects between pentagastrin (low pH) and esomeprazole
(high pH) as regards ghrelin levels point to a physiological role of
ghrelin in the control of gastric acid secretion[27,28]. The
rise in somatostatin concentration is likely due to a direct effect of
the continuous doses of ghrelin, as pH was not affected. The lack of
elevated levels of gastrin for the two groups are probably attributed to
the increase in somatostatin[29] or, although less likely,
low doses of esomeprazole[30,31].

To conclude, the Bravo capsule system is to be used for
prolonged studies of gastric pH in free roaming conscious rats over days
and is well tolerated, and could serve as a complement to the gastric
fistula model, as shown by acid and gut hormone secretion measurements.

ACKNOWLEDGMENTS

The
authors gratefully acknowledge the technical support by Wiveca Ring-Persson
and the invaluable assistance by Berndt Wallin at the Karolinska
University Hospital Solna. This study was funded by GlaxoSmithKline and
the Karolinska Institute.

COMMENTS

Background

The
pharmacological treatment of gastrointestinal acid-related diseases aims
at providing ulcer and mucosal healing, symptom relief and improved
quality of life. Gastric acid inhibitory compounds are widely used in
the clinical setting in order to treat not only benign gastric and
duodenal ulcers, but also gastritis and reflux esophagitis. Over the
past two decades, there has been a number of reports on the use of
proton pump inhibitors (PPIs) such as omeprazole and the following
competitors. The PPIs are activated in the acid environment in the
stomach and inhibit the final step of gastric acid secretion. They bind
in a non-competitive way to the H+, K+ -ATPase and
inhibit secretion. Even though the PPIs have many good properties
compared to other treatment regims, and are considered the treatment of
choice in acid-related gastrointestinal diseases, there are drawbacks
with PPI treatment. For instance, the onset of action is slow as
compared to that of H2-receptor antagonists, which induce an immediate
acid inhibition, and the duration of action may be too short giving room
for night-time acid breakthrough. So far, treatments have got around
this problem by recommending a two-dose regimen. Pharmaceutical
development has been directed against finding a compound with profound
acid inhibitory action over prolonged periods of time, not permitting
night-time acid breakthrough to take place. The development of such
drugs, however, require new methods of studying gastric acid secretion
over prolonged periods, up to 120 h over or more.

Research frontiers

Research
concerning acid-related diseases has been focused on PPIs targeted
against the H+, K+ -ATPase of the stomach and
H2-receptor antagonists. Recent studies have shown that the proton pump
is the most likely candidate for a sustainable therapeutic application
in the regulation of acid suppression. One of the hurdles in this field
is the possibility to perform long-term measurements of acid secretion
in the development of pharmacological treatment of acid diseases.
Although PPIs are highly effective as a class, differences in their
pharmacokinetics, such as bioavailability, metabolism, and elimination
half-life, may translate into differences in clinical outcomes.

Innovations and breakthroughs

Over the latest years, new drugs have emerged on the
market, such as being PPIs (3rd generation), new potassium channel
blocking agents that inhibit gastric secretion (P-CAP), and even
combinations of PPIs and H2-receptor antagonists. A second line to this
further development is to be expected and with this new method,
developed as a tool for evluation of such long-acting drugs, may become
a feasible tool in the clinical setting for treatment of acid-dependent
diseases.

Applications

Our
research demonstrates stable recordings with the Bravo capsule system in
the rat. The animals were given PPI and ghrelin and this resulted in an
almost immediate response in pH, sustained during approximately 6 h. The
capsule model was compared with the fistula model and showed agreement
in compliance between the two methods. This indicates that the capsule
model could eventually replace the fistula model. It seems better to use
the former method because of less strain on the rats, and easier and
more gentle handling and experimental procedures. Furthermore, the Bravo
system set-up is easy to manage and the information recorded allows many
different analysis variables. The system also records over five
consecutive days, which previously has not been possible in this
setting.

Terminology

Bravo
capsule system: A catheter-free system used to measure esophageal pH
(acidity) levels in patients who have or are suspected of having
gastroesophageal reflux disease, but has now also been used for
intragastric titration of pH.

Peer review

The
measurement of intragastric pH with the Bravo capsule system is
comparable to that of the gastric fistula model, and is useful for
prolonged studies of gastric pH, even in free roaming conscious rats
over days, as described. Although further studies are required, this
study indicates the novel possibility for investigating the acid and gut
hormone secretion under more physiological conditions.